Thallium binding to native and radiation-inactivated Na +/K +-ATPase

Abstract

The number of high-affinity K +-binding sites on purified Na +/K +-ATPase from pig kidney outer medulla has been assessed by measurement of equilibrium binding of thallous thallium, Tl +, under conditions (low ionic strength, absence of Na + and Tris +) where the enzyme is in the E 2-forms. Na +/K +-ATPase has two identical Tl + sites per ADP site, and the dissociation constant varies between 2 and 9 μM. These values are identical to those for Tl + occlusion found previously by us, indicating that all high-affinity binding leads to occlusion. The specific binding was obtained after subtraction of a separately characterized unspecific adsorption of Tl + to the enzyme preparations. Radiation inactivation leads to formation of modified peptides having two Tl +-binding sites with positive cooperativity, the second site-dissociation constant approximating that for the native sites. The radiation inactivation size (RIS) for total, specific Tl + binding is 71 kDa, and the RIS for Tl + binding with original affinity is approx. 190 kDa, equal to that of Na +/K +-ATPase activity and to that for Tl + occlusion with native affinity. This latter RIS value confirms our recent theory that in situ the two catalytic peptides of Na +/K +-ATPase are closely associated. The 71 kDa value obtained for total Tl + sites is equal to that for total binding of ATP and ADP and it is clearly smaller than the molecular mass of one catalytic subunit (112 kDa). The Tl +-binding experiments reported thus supports the notion that radiation inactivation of Na +/K +-ATPase is a stepwise rather than an all or none process.